While gene therapy is of great clinical interest for treatment of gene defects, this therapy has not entered into mainstream clinical practice, at least in part because selective delivery of genes to target tissues has proven extremely difficult. Currently, viral vectors are used, particularly retroviruses and adenovirus, which are to some extent selective. However, many vector systems are by their nature unable to produce stable integrants and some also invoke immune responses thereby preventing effective treatment. Alternatively, “naked” DNA is packaged in liposomes or other similar delivery systems. A major problem to be overcome is that such gene delivery systems themselves are not tissue selective, whereas selective targeting of genes to particular tissues would be desirable for many disorders (e.g., cancer therapy). While use of tissue specific promoters to target gene therapy has been effective in some animal models it has proven less so in man, and selective tissue specific promoters are not available for a wide range of tissues.
The current invention has arisen unexpectedly from recent investigations exploring why papillomavirus (PV) late gene expression is restricted to differentiated keratinocytes. In this regard, it is known that PV late genes L1 and L2 are only expressed in non-dividing differentiated keratinocytes (KCs). Many investigators including the present inventors have been unable to detect significant PV L1 and L2 protein expression when these genes are transduced or transfected into undifferentiated cultured cells, using a range of conventional constitutive viral promoters including retroviral long terminal repeats (LTRs) and the strong constitutive promoters of CMV and SV40. PV L1 mRNA can however be efficiently translated in vitro using rabbit reticulocyte cell lysate, suggesting that there are no cellular inhibitors in the lysate interfering with translation of L1. The major difference between the in vitro and in vivo translation systems is that L1 comprises the dominant L1 mRNA in in vitro translation reactions, while it constitutes a minor fraction among the cellular mRNAs in intact cells.
In vivo, PV late proteins are not produced in undifferentiated KC. However, they are expressed in large quantity in highly differentiated KC. The mechanism of this tight control of late gene expression has been poorly understood, and searches by many groups for KC specific PV gene transcriptional control proteins have been unrewarding.
Blockage of translation of L1 mRNA in vivo has been attributed to sequences within the L1 ORF (Tan et al. 1995, J. Virol. 69 5607-5620; Tan and Schwartz, 1995, J. Virol. 69 2932-2945). By using a Rev and Rev-responsive element of HIV, such inhibition could be overcome (Tan et al. 1995, supra). Accordingly, the inventors examined whether removal of putative “inhibitory sequences” in the L1 ORF would allow production of L1 protein in undifferentiated cells. Deletion mutagenesis of BPV L1 to remove putative inhibitory sequences and expression of resultant deletion mutants in CV-1 cells revealed surprisingly that despite expression of L1 mRNA, L1 protein could not be detected.
In view of the foregoing, it has been difficult hitherto to understand how papillomaviruses produce large amounts of L1 protein in the late stage of their life cycle using this apparently “untranslatable” gene. The present inventors have discovered the mechanism by which L1 protein is expressed in the late stage of the life cycle of this virus, and have also discovered a method of general application whereby polynucleotides can be designed or modified in order to effect selective expression of a protein in a target cell or tissue.